摘要
目的观察枸杞多糖(LBP)对2,4-二氯苯氧乙酸(2,4-D)诱导的PC12细胞凋亡蛋白Bax、Bcl-2、caspase-3表达的影响,探讨2,4-D诱导PC12细胞凋亡的可能机制。方法培养PC12细胞,测定细胞生长曲线。分别设对照(无血清培养基)组和400、800、1200μmol/L 2,4-D染毒组及LBP干预组(250 mg/L LBP+1200μmol/L 2,4-D),以CCK-8法检测细胞在不同染毒浓度下的活性变化,以Hochest染色定性分析细胞凋亡情况,以JC-1法测定线粒体膜电位变化,以透射电镜观察染毒后PC12细胞形态变化,以Western blot法检测PC12细胞凋亡相关蛋白Bax、Bcl-2、caspase-3的表达。结果细胞在24~48 h进入对数增长期,边界清晰,胞质透明。与对照组比较,各浓度2,4-D染毒组PC12细胞存活率均降低,ROS含量及凋亡率均升高,差异有统计学意义(P<0.05),且随着2,4-D染毒浓度的升高,PC12细胞存活率呈下降趋势,ROS含量及凋亡率均呈上升趋势。与1200μmol/L 2,4-D染毒组比较,LBP干预组PC12细胞存活率较高,ROS含量及凋亡率均较低,差异有统计学意义(P<0.05)。与对照组比较,各浓度2,4-D染毒组PC12细胞线粒体膜电位均降低,差异有统计学意义(P<0.05),且随着2,4-D染毒浓度的升高,PC12细胞线粒体膜电位呈下降趋势;LBP干预组PC12细胞线粒体膜电位水平升高,差异有统计学意义(P<0.05)。经倒置荧光显微镜观察,染毒组细胞固缩变圆,突触消失。与对照组比较,各浓度2,4-D染毒组PC12细胞Bax、caspase-3蛋白表达量增加,Bcl-2蛋白表达量下降,Bcl-2/Bax比值下降,差异有统计学意义(P<0.05);LBP干预组PC12细胞Bax、caspase-3蛋白表达量下降,Bcl-2蛋白表达量增加,Bcl-2/Bax比值升高,差异有统计学意义(P<0.05)。经透射电镜观察,染毒后400μmol/L 2,4-D组线粒体肿胀,形状不规则呈空泡状,溶酶体被激活;800μmol/L 2,4-D组细胞核膜皱缩,线粒体消失;1200μmol/L 2,4-D组细胞固缩,核膜溶解,可见凋亡小体;LBP干预组细胞膜较完整,轮廓较清晰。结论2,4-D可能通过调节线粒体ROS诱导PC12细胞凋亡,引起PC12细胞Bax、caspase-3蛋白表达增加,Bcl-2蛋白表达量下降,而LBP具有一定的改善作用。
Objective To understand the effects of Lycium barbarum polysaccharide(LBP)on the expression of Bax,Bcl-2 and caspase-3 related to apoptosis of PC12 cells induced by 2,4-D.Methods PC12 cells were cultured and the growth curve was measured,and divided into control group(serum-free medium),400,800,1200μmol/L 2,4-D groups and LPB intervention group(250 mg/L LBP+1200μmol/L 2,4-D).CCK-8 method was used to detect the changes of cell activity,and Hochest staining was used to detect the cell apoptosis.The changes of mitochondrial membrane potential were determined by JC-1 method.The morphological changes of PC12 cells were observed by transmission electron microscopy.Western blot was used to detect the expression of apoptosis-related proteins Bax,Bcl-2 and caspase-3 in PC12 cells.Results The cells grew in logarithmic growth stage after 24-48 h cultivation,which revealed clear boundaries and clear cytoplasm.Compared with the control group,the survival rate of PC12 cells decreased,while the ROS content and apoptosis rate increased significantly in the2,4-D exposure group(P<0.05);With the increase of 2,4-D exposure doses,the survival rate of PC12 cells decreased,while the ROS content and apoptosis rate increased;Compared with the 1200μmol/L 2,4-D group,the survival rate of PC12 cells in the LBP intervention group was higher,while the ROS content and apoptosis rate were lower(P<0.05).Compared with the control group,the mitochondrial membrane potentials of PC12 cells in the 2,4-D exposure groups decreased(P<0.05),and the mitochondrial membrane potential of PC12 cells decreased with the increase of 2,4-D concentration;The mitochondrial membrane potential level of PC12 cells in the LBP intervention group increased(P<0.05).Pyknotic and round PC12 cells and disappearance of synapses were observed in the 2,4-D exposure groups under inverted fluorescence microscope.Compared with the control group,protein expressions of Bax and caspase-3 increased,protein expressions of Bcl-2 decreased,and the ratios of Bcl-2 to Bax decreased in PC12 cells of the 2,4-D exposure groups(P<0.05);Protein expressions of Bax and caspase-3 decreased,protein expressions of Bcl-2 increased,and the Bcl-2/Bax ratio increased in PC12 cells in the LBP intervention group(P<0.05).The observation by transmission electron microscopy showed that the mitochondria were swollen and vacuolated irregularly,and lysosomes were activated in 400μmol/L 2,4-D exposure group;The cell nuclear membrane shrinked and mitochondria disappeared in 800μmol/L 2,4-D exposure group;Cells shrinkage and nuclear membrane dissolution were seen,as well as apoptotic corpuscles in 1200μmol/L 2,4-D exposure group;Intact and clear cell membrane was observed in the LBP intervention group.Conclusion 2,4-D may induce apoptosis of PC12 cells by regulating mitochondrial ROS,leading to the increased expression of Bax and caspase-3 in PC12 cells,the decreased expression of Bcl-2 protein,and LBP may has a certain protective effect on cells.
作者
周健
柴茹
张亚娟
李宏辉
贾磊娜
王凯
朱玲勤
杨惠芳
ZHOU Jian;CHAI Ru;ZHANG Ya-juan;LI Hong-hui;JIA Lei-na;WANG Kai;ZHU Ling-qin;YANG Hui-fang(Department of Occupational Health and Environmental Hygiene,School of Public Health and Management,Ningxia Medical University,Yinchuan,Ningxia 750004,China)
出处
《环境与健康杂志》
CAS
北大核心
2020年第7期605-609,659,共6页
Journal of Environment and Health
基金
宁夏自然科学基金(2018A0101)
